The effects of Crocus sativus (saffron) and its constituents on nervous system: A review

Saffron or Crocus sativus L. (C. sativus) has been widely used as a medicinal plant to promote human health, especially in Asia. The main components of saffron are crocin, picrocrocin and safranal. The median lethal doses (LD50) of C. sativus are 200 mg/ml and 20.7 g/kg in vitro and in animal studies, respectively. Saffron has been suggested to be effective in the treatment of a wide range of disorders including coronary artery diseases, hypertension, stomach disorders, dysmenorrhea and learning and memory impairments. In addition, different studies have indicated that saffron has anti-inflammatory, anti-atherosclerotic, antigenotoxic and cytotoxic activities. Antitussive effects of stigmas and petals of C. sativus and its components, safranal and crocin have also been demonstrated. The anticonvulsant and anti-Alzheimer properties of saffron extract were shown in human and animal studies. The efﬁcacy of C. sativus in the treatment of mild to moderate depression was also reported in clinical trial. Administration of C. sativus and its constituents increased glutamate and dopamine levels in the brain in a dose-dependent manner. It also interacts with the opioid system to reduce withdrawal syndrome. Therefore, in the present article, the effects of C. sativus and its constituents on the nervous system and the possible underlying mechanisms are reviewed. Our literature review showed that C. sativus and its components can be considered as promising agents in the treatment of nervous system disorders.


Introduction
Crocus sativus L (C. sativus), commonly known as saffron, is a small perennial plant belonging to the family of Iridaceas. This plant is cultivated in many countries including Iran, Afghanistan, Turkey and Spain (Abdullaev, 1993). The stigmas of C. sativus are known to contain carotenoids, α-crocetin and glycoside crocin (responsible for saffron yellow color) and picrocrocin, the aglyconesafranal (responsible for saffron aroma) (Fernández and Pandalai, 2004;Champalab et al., 2011), the antioxidant carotenoids lycopene and zeaxanthin and vitamin B2 (Vijaya Bhargava, 2011).
It has been shown that C. sativus stigma aqueous extract and its constituents, crocin but not safranal enhanced the sexual activity in male rats (Hosseinzadeh et al., 2008). Saffron and its constituentscrocin and safranal are also shown to be potent oxygen radical scavengers (Assimopoulou et al., 2005;Mashmoul et al., 2013;Farahmand et al., 2013).
In traditional medicine, C. sativus has been frequently used as an herbal sedative, antispasmodic, aphrodisiac, diaphoretic, expectorant, stimulant, stomachic, anticatarrhal, eupeptic, gingival sedative and emmenagogue (Nemati et al., 2008). C. sativus was experimentally shown to be effective in relieving symptoms of premenstrual syndrome (PMS). Following administration of saffron, a significant effect was observed in cycles 3 and 4 in the Total Premenstrual Daily Symptoms and Hamilton Depression Rating Scale which indicates the efficacy of C. sativus in the treatment of PMS (Agha-Hosseini et al., 2008).
Aqueous (500 mg/kg) and ethanolic extracts of C. sativus petals reduced blood pressure in a dose-dependent manner in rats (Fatehi et al., 2003). Administration of the aqueous extract of saffron petals (500 mg/kg) reduced blood pressure from 133.5±3.9 to 117±2.1 mmHg in rats. This reduction was postulated to be due to the effect of the extracts on the heart itself, total peripheral resistance or both (Fatehi et al., 2003). In rats isolated vas deferens, contractile responses to electrical field stimulation (EFS) were decreased by the petals extracts (Fatehi et al., 2003). EFSinduced contractions of vas deferens were shown to be mediated by noradrenaline and adenosine triphosphate (ATP) released as co-transmitters from sympathetic nerves (Hoyle and Burnstock, 1991). The ethanolic extract made more pronounced changes in EFS in rats isolated vas deferens whereas in guinea pig ileum, the aqueous extract of the plant was more effective (Fatehi et al., 2003). Crocin analogs isolated from saffron remarkably increased the blood flow in the retina and choroid and facilitated retinal function recovery; therefore, it could be used to treat ischemic retinopathy and/or agerelated macular degeneration (Xuan, 1999). One study suggested that saffron exerted a significant cardioprotective effect by preserving hemodynamics and left ventricular functions (Sachdeva et al., 2012). Administration of C. sativus extractinpatients who had normal white blood cells (WBC) count, significantly increased WBC compared to crocin or placebo. Moreover, other hematologic factors were not changed significantly during 3 months of the study (Mousavi et al., 2015).
An in vitro study showed the inhibitory activity of saffron and crocin on amyloid beta-peptide fibrillogenesis and its protective action against H 2 O 2 -induced toxicity in human neuroblastoma cells (Papandreou et al., 2006(Papandreou et al., , 2011. Additionally, administration of saffron (60 mg/kg body weight, i.p.) to normal and aged mice for one week, significantly improved learning and memory (Papandreou et al., 2011). Also, in vitro studies have confirmed the neuroprotective effects of saffron and its constituents in amnesic and ischemic rat models (Hosseinzadeh and Sadeghnia, 2005;Ochiai et al., 2007).
Considering clinical and animal experimental studies, the present review explores the important effects of C. sativus and its constituents on nervous system.

Methods
Information of this review article was collected by searching for the key-words "Crocus sativus", "nervous system", "clinical application", "animal studies", "crocin", "crocetin" and "safranal" in databases namely ISI Web of Knowledge, Medline/ Pubmed, Science direct, Scopus, Google Scholar, Embase, Biological Abstracts and Chemical Abstracts.
Crocetin isolated from saffron is one of the two principal chemicals responsible for the red color of saffron (Martin et al., 2002). Crocetin constitutes approximately 0.3% of the total weight of the saffron stigma (Escribano et al., 1996, Dris andJain 2004). Crocetin can function as an acid (anionic) dye for biological staining because it has a carboxyl group at each end of the polyene chain which is easily dissolved in aqueous alkali solutions at pH ≥ 9. Crocetin is mostly present as trans isomer but cis-crocetin and its glycosides are also present in saffron as minor components (Melnyk et al., 2010).
Crocin belongs to a group of natural carotenoid commercially obtained from the dried stigma of C. sativus. It has a deep red color, forms crystals with a melting point of 186 o C and is easily soluble in water. Crocin is responsible for the color of saffron. Structure of crocin was elucidated by Karreeet al (1935). It is the main pigment of saffron (approx. 80% of pigment content). Pure crocin can be isolated from saffron extract and is directly crystallized (Karrer et al., 1932). Crocin is not orally absorbed. Crocins are hydrolyzed to crocetin before or during intestinal absorption, and the absorbed crocetin is partly metabolized to mono and diglucuronide conjugates (Asai et al., 2005).
Safranal (which is fat soluble) and pigments of the crocetin carotenoid are bitter, but the most important cause of saffron bitterness is picrocrocin (Abdullaev, 1993). Saffron lipophilic carotenoids are lycopene, alpha-and betacarotene and zeaxanthin (Winterhalter and Straubinger, 2000;Tarantilis and Polissiou, 1997). Kaempferol has also been found in alcoholic extract of saffron petals (Gregory et al.,2005). Flavonoids especially lycopene, amino acids, proteins, starch, resins and other compounds have also been shown to be present in saffron (Assimopoulou et al.,2005). Saffron also has trace amounts of thiamine and riboflavin (Alonso et al.,2001).

Anticonvulsant effects
In Iranian folk medicine, C. sativus had been used as an anticonvulsant herb (Khosravan, 2002). Experimental studies also confirmed saffron anticonvulsant effects in rats and mice (Sunanda et al., 2014;Khosravan, 2002). Saffron at the doses of 400 and 800 mg/kg showed a significant antiepileptic activity in pentylenetetrazole (PTZ)-induced seizure model in a dose-dependent manner. However, saffron at the dose of 200 mg/kg did not significantly suppress PTZinduced seizures (Sunanda et al., 2014). The anticonvulsant activities of aqueous and ethanolic extracts of saffron have been demonstrated in mice using maximal electroshock seizure (MES) and PTZ models (Khosravan, 2002).

Anti-Alzheimer effects Basic studies
Alzheimer's disease (AD) is described pathologically as deposition of amyloid βpeptide (Aβ) fibrils. The aqueousethanolic (50:50, v/v) extract of C. sativus stigmas has good antioxidant properties -higher than those of carrot and tomato-in a concentration and time-dependent manner which was accompanied by inhibition of Aβ fibrillogenesis. The transcrocin-4, the digentibiosyl ester of crocetin was the main carotenoid constituent which inhibited Aβ fibrillogenesis (Papandreou et al., 2006). Intracerebroventricular (ICV) injection of streptozotocin (STZ) to rodents has been frequently used as an animal model for sporadic AD (Lannert and Hoyer, 1998;Labak et al., 2010;Veerendra Kumar and Gupta, 2003). It has been previously revealed that treatment by C. sativusextract (30 mg/kg) for 3 weeks could significantly improve cognition deficits induced by ICV injection of STZ in rats (Khalili et al., 2010).Crocin (30 mg/kg) has also been shown to have an antagonizing effect on the STZ-induced cognitive deficits in rats (Khalili and Hamzeh, 2010).
Geromichaloset al. (2012) showed that the saffron extract had a moderate (up to 30 %) inhibitory activity on acetylcholinesterase (AChE) and inhibited acetylcholine breakdown which is the main therapeutic approach for AD (Geromichalos et al., 2012).

Clinical studies
Administration of saffron 30 mg/day (15 mg twice daily) was found to be as effective as donepezil for treatment of mild-to-moderate AD in the subjects of 55 years and older (Akhondzadeh et al., 2010a ). In addition, the frequency of saffron extract side effects was similar to those of donepezil except for vomiting, which occurred more frequently in the donepezil group (Akhondzadeh et al., 2010a). In another study, 46 patients with mild-to-moderate AD were treated by saffron for 16 weeks. The results showed that the cognitive functions in saffrontreated group were significantly better than placebo (Akhondzadeh et al. 2010b).

Basic studies
Crocin and ethanolic extracts of saffron are known to have antidepressant effect in rodents. Using forced swimming test, it was shown that crocin (50-600 mg/kg) reduced immobility time while increased climbing time (Hosseinzadeh et al., 2003). In other studies,effectiveness of antidepressant activity of C. sativus extract was described (Karimi et al., 2001;Yang Wang et al., 2010). The petroleum ether and dichloromethane fractions were suggested to be the active parts of corms of C. sativus. The petroleum ether fraction of the extract of C. sativus L. corms mainly contained n-tridecane, ntetradecane, n-pentadecane, diethyltoluamide, n-catane and nheptadecane, etc. (Yang Wang et al., 2010).
Kaempferol, a C. sativus petal constituent also reduced immobility behaviors in mice (100 and 200 mg/kg) and rats (50 mg/kg) (Hosseinzadeh et al., 2007). A decreased time of immobility in rodents caused by selective serotonin reuptake inhibitors such as fluoxetine may explain the antidepressant effects of the plant (Cryan and Lucki, 2000;Lucki, 1997). The antidepressant effect of aqueous and ethanolic extracts of C. sativus petal and stigma has been shown in mice (Karimi et al., 2001). Major constituents of saffron, safranal and crocin, also had antidepressant activity in mice (Hosseinzadeh et al., 2004).
The effectiveness of C. sativus as a treatment for depression in animal model was shown in Table 1.

Clinical studies
In a randomized and double-blind clinical trial study, saffron supplementation statistically improved the mood of subjects compared to the placebo group. For six weeks, 30 mg/day of saffron was given and subjects were evaluated based on the Hamilton Depression Rating Scale (HAM-D) (Akhondzadeh et al., 2005). Another similar study by Noorbala et al. (2005) revealed that six-week administration of saffron extract (30 mg/day) was effective in the treatment of mild to moderate depression. These effects were similar to the effects of fluoxetine (Noorbala et al., 2005) and imipramine 100 mg/day (Akhondzadeh et al., 2004). Therapeutic benefits of petals of C. sativus in the treatment of mild to moderate depression have also been suggested (AkhondzadehBasti et al., 2007). The efficacy of co-administration of hydroalcoholic extract of C. sativus (40 or 80 mg) and fluoxetine (30 mg/ day) was also investigated in a double-blind randomized clinical trial for six weeks. The results revealed that a dose of C. sativus 80 mg plus fluoxetine was more effective than that of C. sativus 40 mg and fluoxetine to treat mild to moderate depressive disorders (Moosavi et al., 2014).
Table1. The effectiveness of C. sativus as a treatment for depression in animal models. Short-term administration of saffron (30 mg/day) capsules for six weeks was also shown to be as effective as fluoxetine (40 mg/day) in improving depression symptoms in patients who were suffering from major depressive disorder (MDD) after undergoing a percutaneous coronary intervention (Shahmansouri et al., 2014).

Aqueous and ethanolic extract
Another clinical study demonstrated that saffron aqueous extract (15 mg twice daily) andcrocin (15 mg twice daily) were well tolerated by patients with schizophrenia during the study and no serious side effects were observed (Mousavi et al., 2015).
The effectiveness of C. sativus as a treatment for depression in human studies was summarized in Table 2.

Anti-Parkinson effects
Saffron and its components (mainly crocin, crocetin, and safranal) have been used in animal models with neurodegenerative diseases (Ochiai et al., 2007;Purushothuman et al., 2013). Crocin and safranal have inhibitory effect on fibrillation of apo alpha-lactalbumin (aalpha-LA), under amyloidogenic conditions which crocin was found to be more effective than safranal. Formation of toxic amyloid structures is related with various neurodegenerative diseases such as Alzheimer's and Parkinson's diseases (Ebrahim-Habibi et al. 2010).
Neuroprotective effects of seven-day administration of crocetin (25, 50 and 75µg/kg body weight, i.p.) against 6hydroxydopamine (6-OHDA, 10 µgintrastriatal)-induced Parkinson's disease in rats have been reported. Reduction in dopamine utilization by tissues was suggested as a possible mechanism (Ahmad et al., 2005). In another study, the protective effect of saffron pre-treatment on dopaminergic cells in the substantia nigra pars compacta (SNc) and retina in a mouse model of acute MPTP (1-methyl-4-phenyl-1,2,3,6tetrahydropyridine)-induced Parkinson's disease was examined. BALB/c mice received MPTP or saline over a 30-hour period. Animals in the saffron-treated group received Saffron (0.01% w/v) dissolved in the drinking water for five days and control groups received normal tap water. After the six days, the brains were processed for tyrosine hydroxylase (TH) immunochemistry and TH+ cells count was reported using the optical fractionator method. In both the SNc and retina, the MPTP-injected mice had a reduced number of TH+ cells (30-35%) compared to saline-injected controls. Pretreatment of MPTP-injected mice by saffron increased both SNc and retinal TH+ cell counts (25-35%) and closed them to the control levels. It was concluded that saffron pre-treatment saved many dopaminergic cells in the SNc and retina from Parkinsonian (MPTP) insult in mice (Purushothuman et al., 2013).

Effects of C. sativus on oxidative damages and neurotoxicity
It has been reported that crocin 10 μM inhibited the formation of peroxidized lipids in cultured PC12 cells, moderately restored superoxide dismutase (SOD) activity and maintained neurons morphology. While the antioxidant effect of crocin was comparable to that of αtocopherol, it was even more pronounced at some concentrations.
Administration of C. sativus stigma extract (100 mg/kg, p.o.) for 7 days before induction of cerebral ischemia by middle cerebral artery occlusion (MCAO) remarkably reduced SOD, catalase and Na, K-ATPase activities and glutamate and aspartate concentrations induced by ischemia in rats (Saleem et al. 2006). Treatment with saffron extract (5 and 25 mg/ml) and crocin (10 and 50 μM) could decrease the neurotoxic effect of glucose in PC12 cells. The results showed that glucose (13.5 and 27 mg/ml) reduced PC12 cells viability while cell death was reduced by saffron and crocin pretreatment (Mousavi et al., 2010). Another study showed that administration of saffron extract (200 mg/kg) and honey syrup (500 mg/kg) for 45 days reduced the aluminium chloride-induced neurotoxicity in mice (Shati et al. 2011). Other studies showed that safranal has some protective effects on different markers of oxidative damage in hippocampal tissue from ischemic rats (Hosseinzadeh and Sadeghnia, 2005) and in hippocampal tissue following quinolinic acid (QA) administration (Sadeghnia et al., 2013). Safranal also reduced extracellular concentrations of glutamate and aspartate (excitatory amino acids) in the hippocampus of anaesthetized rats following kainic acid administration (Hosseinzadeh et al. 2008b).
In addition, crocin increased the activity of SOD and glutathione peroxidase (GPx) and remarkably reduced malondialdehyde (MDA) content in the ischemic cortex in rat model of ischemic stroke (Vakili et al., 2013). Co-administration of saffron extract with aluminium reversed aluminiuminduced changes in monoamine oxidase (MAO-A, MAO-B) activity and the levels of lipid peroxidation in whole brain and cerebellum (Linardaki et al., 2013).
It has been suggested that exposure to high levels of glucocorticoids or chronic stress may lead to oxidative injury in the hippocampus, which may impair learning and memory functions (Behl et al., 1997;McIntosh et al., 1998a). Saffron extract and crocin can improve learning and memory (Abe andSaito, 2000, Pitsikas et al., 2007). It was demonstrated that saffron and crocin can prevent oxidative stress in the hippocampus and avoid deficits in spatial learning and memory (Ghadrdoost et al., 2011). It has been reported that crocetin increases the antioxidant potential in brain and helps to fight against 6-OHDA-induced neurotoxicity (Ahmad et al., 2005).
Table2. The effectiveness of C. sativus as a treatment for depression in human studies.

Effects of C. sativus on neuronal injury and apoptosis
Crocin (30, 60 and 120 mg/kg) showed protective effect against ischemia/reperfusion injury and cerebral edema in a rat model of stroke and decreased infarct volume. Administration of crocin (60 mg/kg), one hour before, or one hour after the induction of ischemia, reduced brain edema (Vakili et al., 2013). The neuroprotective effects of crocetin in the brain injury in animal studies have been suggested to be related to its ability to inhibit apoptosis at early stages of the injury and its ability to promote angiogenesis at the subacute stage as directed by higher expression levels of vascular endothelial growth factor receptor-2 (VEGFR-2) and serum response factor (SRF) (Bie et al., 2011).
In serum/glucose-deprived cells, lipid peroxidation may increase which can be inhibited by crocin. Crocin can suppress the activation of caspase-8 was and its antioxidant properties are more prounounced than α-tocopherol at the same concentration (Ochiai et al., 2004). In addition, crocin suppressed the activation of caspase-8 caused by serum/glucose deprivation (Ochiai et al., 2004a). Crocin and tricrocin remarkably suppressed membrane lipid peroxidation, caspase-3 activation and cell death in serum-deprived and hypoxic PC12 cells which were more marked than those of tricrocin. Crocetin has been suggested to have some linked glucose esters (Ochiai et al., 2007). The results of this study suggested that dicrocin and picrocrocin had no effect on cell survival (Ochiai et al., 2007).
Endoplasmic reticulum (ER) stress has been shown to be closely related to inflammatory pathways (Mori, 2009). It was shown that EAE increases the transcript levels of the ER stress genes XBP-1/s (Marciniak et al., 2004). Administration of crocin on day 7 post-EAE induction, suppressed ER stress and inflammatory gene expression in the spinal cord and also reduced the expression of ER stress genes XBP-1/s (Deslauriers et al., 2011).

C.
sativus and the brain neurotransmitters Ettehadi et al. (2013) showed that the aqueous extract of saffron (50, 100, 150 and 250 mg/kg, i.p.) increased brain dopamine concentration in a dosedependent manner. Moreover, the extract had no effect on brain serotonin or norepinephrine concentration. In addition, the results showed that the aqueous extract of saffron especially at the dose of 250 mg/kg triggered and increased the production of important neurotransmitters including dopamine and glutamate in rat brain (Ettehadi et al., 2013).
The effects of saffron on conditioning place preference (CPP) induced by morphine has been reported to be similar to the effect of N-methyl-D-aspartate (NMDA) receptor antagonists (Hosseinzadeh et al., 2012Lechtenberg et al., 2008. Furthermore, the analgesic effect of saffron can be reduced by NMDA receptor antagonists (Nasri et al., 2011). Therefore an interaction with glutamatergic system for saffron of its components might be postulated.
The NMDA receptors have also been well known to be involved in post-training memory processing by the amygdala and hippocampus (Izquierdo et al., 1992). The role of these receptors in morphine statedependent learning has also been suggested (Zarrindast et al., 2006;Cestari and Castellano, 1997). Involvement of NMDA receptors in the effects of C. sativus or its constituents on memory has been shown (Lechtenberg et al., 2008;Abe et al., 1999). The beneficial effects of saffron on memory have also been suggested to be mediated by the cholinergic system (Pitsikas and Sakellaridis, 2006;Ghadami and Pourmotabbed, 2009).
Intraperitoneal administration of ethanolic extract of saffron (10, 50 and 100 mg/Kg) and safranal (1, 5 and 10 mg/Kg) reduced theacquisition and expression of morphine CPP (Ghoshooniet al., 2011). Administration of crocin (400 and 600 mg/kg,i.p.) 30 min before morphine administration decreased the acquisition and reinstatement of morphineinduced CPP in mice (Imenshahidi et al., 2011). It has also been reported that 5 min after morphine (10 mg/kg) administration, injection of ethanolic extract of C. sativus stigma (5 and 10 µg/rat) into the nucleus accumbens shell part of rats, led to decrease in the time spent in drug paired side. In addition, injection of extract to the animals that received morphine (10 mg/kg), decreased the expression of morphine (CPP) (Mojabi et al., 2008). Injection of aqueous extract of saffron stigma (50, 100, 150 and 250mg/Kg,i.p.) showed an increased release of dopamine in rat brains. Also, this extract (only at 250 mg/Kg) significantly increased the release of glutamate (Ettehadi et al., 2013).
Administration of saffron extract (150 and 450 mg/kg) before retention trials also increased the time latency. So, saffron extract reduced morphine-induced memory impairment (Naghibi et al., 2012). Protective effect of saffron extract against morphine-induced inhibition of spatial learning and memory in rat has also been suggested (Haghighizad et al., 2008).
The effects of C. sativus on opioid receptors were showed in Table 3.

Conclusion
Anti-oxidant and anti-inflammatory effects of the extracts of C. sativus and its constituents (crocetin, crocins, safranal) implies saffron therapeutic potential for various nervous system disorders. Based on the literature, beneficial effects of the plant and its components on neurodegenerative disorders such as Alzheimer and Parkinson's disease are mainly due to their interactions with cholinergic, dopaminergic and glutamatergic systems. It is assumed that saffron anticonvulsant and analgesic properties and its effects on morphine withdrawal and rewarding properties of morphine might be due to an interaction between saffron, GABA and opioid system.
According to human and animal studies, saffron and its constituents have been shown to be effective in the treatment of mild to moderate depression which may be because of an interaction with the serotonin and noradrenaline system. However, to have a detailed perspective of saffron effects on nervous system, more mechanistic investigations are highly advised.

Conflict of interest
There is no conflict of interest.